1. Field of the Invention
The present invention relates to isolated viable parasite cells and to uses thereof. In particular, the present invention relates to vaccines for prevention or treatment of parasitic infestation or infection utilizing immunogenic material derived from isolated viable parasite cells.
2. Background Art
Parasite infections are widespread in the equine and livestock production industries. Losses attributed to parasitism are estimated in the hundreds of millions of dollars (Gibbs and Herd, 1986). Parasitism may manifest as either a clinical or subclinical condition. Although clinical parasitism may appear in a more dramatic fashion, subclinical parasitism is more pervasive, causing declines in feed efficiency, reproductive function and susceptibility to disease. These effects are complicated by the age of the animal, types of parasites present, nutritional and environmental stresses, management systems, presence of other disease conditions, genetic histories and numerous other factors (Gibbs and Herd, 1986; Hawkins, 1993).
Control of parasitism in the United States has centered on the use of anthelmintics, with literally billions of dollars spent annually on the administration of such products (Lanusse and Prichard, 1993). Traditionally, control has been therapeutic and curative in nature; animals are treated to prevent death rather than infection. Serious disease and mortality is decreased, but subclinical losses between treatments persist as a result of reinfection from contaminated pastures or stalling areas.
More recently, a more preventative approach to nematode control has become popular, depending on either strategic treatment with antheimintics alone, or in combination with grazing/pasture management practices (Williams, 1986; Miller, 1993; Stromberg and Corwin, 1993). Rather than concentrating on adult parasites, the aim of these programs has been to decrease pasture contamination with infective larvae, thereby reducing the risk of parasite exposure. This in turn reduces the effects of subclinical parasitism within a livestock herd. Although anthelmintics afford many economic advantages, their use carries some distinct disadvantages; notably development of resistance and the potential hazards of persistent residues and ecotoxicity (Waller, 1993).
Complicating the efficacy of anthelmintics is the development of resistance, which has been well documented in ruminants. It was first reported in 1957 when Haemonchus contortus was found to be resistant to phenothiazine (Drudge, 1957). Resistance continues to be a problem even with the newer classes of anthelmintics. Although it is primarily a problem in horses and small ruminants, some resistance has also been reported in cattle parasites. Resistance of Ostertagia to levamisole (Lyons, 1981; Geerts, 1987; Williams, 1991; Williams, 1991) and to sustained release boluses of morantel (Borgsteede 1988), with side-resistance to levamisole (Borgsteede 1991), and resistance of Trichostrongylus axei and Cooperia oncophora to oxfenbendazole (Eagleson & Bowie, 1986; Jackson, 1987) have been documented. Anthelmintic resistance occurs with all classes of drugs used to control nematodes. Cross-resistance, multiple resistance and side resistance have been reported (Craig, 1993). Development of resistance is believed to be encouraged by rapid "rotation" between different preparations. Reversion or selection away from resistance, once the selection pressure is removed, is slow (Kelly & Hall, 1979).
Development of vaccines against gastrointestinal parasites of cattle has in general produced less than optimal results. Because ruminant gastrointestinal nematodes thrive irrespective of the immune system, a vaccine mimicking this immunological equilibrium is unlikely to be of high efficacy. Vaccines capable of inducing protection via a mechanism different from that mimicking natural immunity would theoretically be more successful (Willadsen, 1993).
Because of their physical location, "concealed" antigens from gut tissue are not normally exposed or "visible" to the host's immune system and therefore do not normally elicit an immune response. Vaccination of the host with isolated preparations of "concealed" antigens from various parasites has shown some potential in inducing a lethal immune response.
Gut tissue from the Anopheles mosquito was first used as a source of antigen for the production of a vaccine. Mosquitoes that took blood meals from rabbits injected with homogenates of heterologous cell fractions from mosquito midgut had a higher death rate than those fed on control rabbits (Alger & Cabrera, 1972). Cattle and guinea pigs were immunized with homogenates of heterologous cell fractions containing antigens extracted from the gut of partially fed Dermacentor andersoni ticks. Engorgement and egg production were significantly reduced in ticks that fed on vaccinated animals (Allen & Humphreys, 1979). Similar success was achieved with calves vaccinated with Amblyomma americanum (McGowan, 1981). These successes and the emergence of acaricide-resistant strains of ticks encouraged work on the cattle specific tick, Boophilus microplus.
Immunization of cattle with crude extract of partially fed ticks decreased tick populations (Johnston, 1986). This protection was different from the naturally acquired resistance that involves a hypersensitivity reaction at the site of tick attachment, which is a reaction not present in the response to immunization (Kemp, 1986). Histopathology of gut tissue from ticks fed on immunized cattle showed damage not evident in ticks fed on cattle with natural tick infestations (Agbede & Kemp, 1986). In addition, cattle injected with crude tick gut membrane and adjuvant had significantly higher antibody levels than naturally infested cattle. Cattle vaccinated with crude gut membrane antigen and then challenged with parasites did not display any obvious anamnestic response, although the challenge dosage was sufficient to produce a significant, but low antibody response in naive animals (Opdebeeck & Daly, 1990). These observations support the contention that vaccination with gut membrane and natural tick infestation do not invoke the same immune response.
Experiments done with purified crude tick extract demonstrated that the immunoprotective antigen was associated with parasite gut membrane (Opdebeeck, 1988; Willadsen, 1988; Willadsen, 1989). Further characterization of the antigen revealed it to be a membrane-bound glycoprotein referred to as Bm86. Immunization of host animals with this antigen decreased tick survival, engorgement weights and fecundity. Antibody to this antigen rapidly inhibited the endocytotic activity of parasite digestive cells (large lumen side gut cells separated from the basement membrane) in the tick gut. This antigen was cloned and expressed as inclusion bodies in E. coli. Ticks fed on cattle vaccinated with these inclusion bodies were significantly damaged, but not killed (Rand, 1989).
Monoclonal antibodies, produced against Boophilus microplus midgut membrane precipitated antigens, were &gt;99% protective in challenge studies. These antigens separated into one major and five minor bands upon the application of conventional SDS-PAGE, indicating that the epitope recognized by the monoclonal antibody is repeated on several antigens. These antigens are thought to be different from Bm86 because vaccination with these antigens results in tick death (Lee & Opdebeeck, 1991).
Antigens may be common to more than one stage of the parasite life-cycle and the shared reactive epitopes may occur on different proteins in the different stages (Maizels, 1987). Because antibody levels have been correlated to the level of protection provided by immunization with tick gut antigen (Opdebeeck, 1988; Lee & Opdebeeck, 1991), larval and adult antigen extracts were purified using anti-gut antibodies. Protection provided by both larval and adult purified antigens was greater than 80%, thus the protective antigens may be common to both stages. Extracts from tick egg membrane were found to be immunogenic, but not protective, to challenge infections. Anti-egg membrane and anti-gut membrane antibodies were cross reactive, recognizing common antigens for the egg and tick gut (Kimaro, 1993).
Because anti-tick antibodies in the sera of cattle vaccinated with tick gut membrane and of cattle naturally infested with ticks reacted with adult tick salivary gland and gut antigens as well as with larval antigens, it was thought that Boophilus microplus gut antigens were not truly "concealed" antigens (Opdebeeck & Daly, 1990). It was determined, however, that when antisera from naturally infested cattle reacted with Bm86, it was through a cross-reactive carbohydrate epitope which had no deleterious effect on ticks (Willadsen & McKenna, 1991). Thus, the gut antigen Bm86 is "concealed" and its polypeptide epitopes are responsible for providing immunoprotection.
"Concealed" antigens have also been proposed as a means of controlling cat flea infestations involving species of Ctenocephalides feli. Imunoglobulins produced in rabbits immunized with homogenates of cell fractions containing antigens from the midgut of fleas were fed to cat fleas and shown to have harmful effects. Dogs immunized with crude antigens and challenged with fleas had fewer surviving fleas than did control animals and surviving females laid fewer eggs (Heath, 1994).
Species of Haemonchus contortus, an economically important blood feeding nematode in sheep, has also been the target of vaccine development. Nonspecific immune responses induced by injections of Freund's complete adjuvant provided some protection against Haemonchus contortus (Bautista-Garfias, 1991). Vaccination with cuticular collagen was not protective although it was immunogenic (Boisvenue, 1991). In contrast, soluble antigens from adults and third stage larvae proved to be poor immunogens (Cuquerella, 1991).
Contortin is an extracellular, polymeric protein which is loosely associated with the lumenal surface of the nematode gut epithelium plasma membrane. Vaccination with a contortin-rich extract prepared from whole worm homogenates is protective in young lambs. Nematode populations in vaccinated animals are smaller in numbers than those found in control animals (Munn, 1987). Serum antibodies precipitated several components of the contortin-rich extract.
Vaccination with crude extracts of gut tissue from adult nematodes and third stage (L3) larvae provided similar protection in goats (Jasmer & McGuire, 1991). Reductions both in numbers of worms and in egg output were achieved in the immunized group. Antibodies from immune serum recognized seven gut proteins, some of which were integral membrane proteins. This antigen preparation may contain a significant amount of contortin (Munn, 1993b). Immunohistochemistry provided confirmation that the antigen originated from parasite intestinal cell populations and demonstrated cross-reactivity with microvillar proteins in Ostertagia ostertagi and several equine small strongyles. Reduction in the number of nematodes recovered after immunization with Haemonchus contortus gut extract was confirmed by Smith (1993) in young Suffolk lambs. Serum from sheep exhibiting natural immunity to Haemonchus contortus did not react with the gut membrane proteins, confirming the "concealed" nature of these proteins. Passive transfer with immune serum from vaccinated sheep decreased egg output in recipient animals. The presence of host antibody coating the microvilli of nematodes recovered from these animals suggested antibody as the effector mechanism. No lesions were observed in the gut membranes. Coating of the microvilli may neutralize necessary proteins (i.e. enzymes) resulting in the death of the worm, or the coating may mechanically block nutrient absorption, effectively starving the nematode.
Antigen H11, present in both fourth (L4) and fifth-stage (L5) larvae of Haemonchus contortus, is the major microvillar integral membrane protein of Haemonchus contortus. Vaccination of young Merino lambs with H11 and with an H11 enriched preparation (containing a small amount of peripheral membrane protein, P1) resulted in a reduction in mean number of nematodes and nematode egg output. Late onset of egg production was noted, suggesting that the effector mechanism may act on pre-adult stages of the parasite (Munn, 1993a). Reductions in numbers of worms and egg output correlated with the serum antibody titer to H11 (Smith, 1993; Tavernor, 1992a,b). The enzymatic nature of H11 has been deduced from DNA sequencing and confirmed by assay and specific inhibitor studies (Munn, 1993). The activity of H11 is inhibited by serum antibodies from vaccinated animals. Most of the antibodies produced are targeted at H11 (Munn, 1993) and levels of inhibition correlate with levels of protection (Munn, 1993). Like contortin, host immunoglobulin appears to be the effector mechanism. Host antibody binds to the parasite intestine as early as seven days post-infection, with lethality observed in nematodes between days 7 and 14. Larvae younger than day 7 post-infection are apparently not susceptible to the immune response. Antigen H11 immunized lambs challenged with trickle inoculations were largely protected against the anemia and egg output observed in challenge controls. They grew as efficiently as the uninfected controls and acquired natural immunity during the course of the trickle infection. Animals challenged with either benzimidazole-resistant or susceptible strains of Haemonchus contortus were equally protected by H11 vaccination (Smith & Smith, 1993). Female parasites were lost more quickly than males, accounting for the reduction in egg output.
Vaccination with fractions of the whole worm H11 enriched extract showed the protective activity to be associated primarily with H11. Another fraction, P1 or H45 was also protective but in much greater amounts than H11. Immunization with H11 enriched extract (containing P1 ) conferred protection in Dorset lambs and Clun Forest sheep (Tavernor, 1992a; Munn, 1993b), but greater nematode reduction was observed in Clun Forest sheep. This difference in protection could be due to breed, quantity of antigen, or age of lambs. In a direct comparison of the protection conferred by vaccination with H11 enriched contortin-free antigen (Munn, 1993b) and by vaccination with contortin-enriched antigen (Munn, 1987), the mean protection (i.e. worm number and egg production declines) achieved with the H11 enriched contortin-free preparations was equal to the best protection achieved with the contortin-enriched preparations, even though lesser amounts of H11 protein were used. Thus, H11 is more effective than contortin. Sufficient protection was achieved with immunization using 100 mg of H11 antigen and greater protection was not demonstrated with larger doses of antigens (Tavernor, 1992a). Vaccination with 95% pure H11 reduced the number of nematodes up to 93% with a 94.6% reduction in egg production.
Cross protection was not demonstrated in challenge studies with Ostertagia circumcincta and Nematodirus battus. This may be a reflection of antigenic difference or because nematode ingestion of host imunoglobulin was in amounts insufficient to promote lethal injury (Smith, 1993). Monoclonal antibodies made against gut surface epitopes of Haemonchus contortus identified epitopes also located in the body wall, the region of the cuticle and on internal organs of third-stage (L3) larvae as well as in the gut and tissues of Ostertagia ostertagi, Trichostrongylus colubriformis, equine small strongyles and Caenorhabditis elegans (Jasmer, 1992).
Another protective component was isolated from the integral membrane fraction of intestinal cells using lectins as ligands to purify the microvillar glycoproteins from whole worm extracts. This fraction, Haemonchus galactose containing glycoprotein complex or H-gal-GP is readily separated from H11 or P1 by SDS-PAGE, its lectin binding specificity and its lower isoelectric point. In a side by side comparison study, H-gal-GP was less protective than H11 and reductions in numbers of nematodes were not as great as for H11, although reductions in egg production were similar. Like H11, H-gal-GP is more effective against female worms than male worms. Comparisons in the literature show H-gal-GP to be more effective than the H45 complex. Differences in protection induced by H-gal-GP and H45 may be due to the specific immunization protocol used.
The establishment of a heterogenous cell line from a plant parasite (caterpillar stage) has been described (Manousis & Ellar, 1990). These authors stated that this was the first time such a technique had successfully been performed with a nematode. Heterogenous (non-specific) cell populations survived for no longer than three months. Supplementation of growth medium with fetal bovine serum (FBS, 10% v/v) supported propagation of cell populations for a period of just greater than five months. Kurti et al. (1988) describe the propagation of heterogenous cell lines derived from tick (Dermacentor variabilis, Rhipicephalus appendiculatus, Rhipicephalus sanguineus, and Boophilus microplus) embryos in growth medium containing 10% FBS, although there was no attempt to propagate a specific cell line through selective laboratory techniques. In another description of parasite cell line propagation, whole parasites were homogenized as staring material, so no attempt was made to selectively cultivate a specific cell line (Hobbs et al., 1993). Parasite homogenate was transferred to wells of a tissue culture plate containing a buffalo rat liver "feeder" cell layer and serum "free" growth medium was utilized. Modification of a DMEM-like growth medium to contain less KC1 and glucose allowed maintenance of viable cell lines for four weeks or longer. Planting of juvenile worm cells on a feeder layer of irradiated buffalo rat liver (BRL) cells extended the viability of cell clusters from a few weeks to as long as six months. Feeder layers of bovine endothelial or mouse embryo (3T3) cells were less effective. Kurtti and Munderloh (1984) described production of mosquito cell culture from larval tissues, adult ovary and embryonic tissues, resulting in the cultivation of heterogenous mixtures of mosquito cells for several years. Munderloh et al. (1994) describe the propagation of heterogenous cell populations from embryonated tick eggs. These investigators described difficulty in preserving the cells in liquid nitrogen for long periods of time. Cells were propagated in tissue culture growth medium containing fetal bovine serum (FBS 20% v/v). The time interval between initiation of the primary culture and the first subculture ranged from 6 to 12 months.
As can be seen from the studies described above, antigens expressed by parasite cells have shown potential in providing protective immunity in sheep and cattle. Unfortunately, these fractions were derived from parasite intestinal tracts which have been harvested manually by microdissection. Thus, it is very labor intensive and expensive to obtain sufficient amounts of immunogenic proteins. Further, it is difficult to obtain sufficient purity of antigens and to identify antigens that may be useful in vaccines. Cell lines which have been established from parasites have been heterogenous and undifferentiated populations which have been difficult to sustain in a tissue culture environment for extended periods of time. Thus, a need exists for homogenous populations of parasite cells that can be sustained in culture for prolonged periods of time.
The present invention fulfills this need by providing homogenous populations of parasite cells which are sustainable in culture for prolonged periods of time and methods for producing such homogenous populations.